The differentiated function of the thyroid cell is regulated in large measure by thyrotropin, also known as thyroid stimulating hormone (TSH) Receptor-mediated effects of TSH on adenylate cyclase activity, with eventual regulation of iodoprotein metabolism, are generally accepted Receptor-mediated effects of TSH on electrochemical ion gradients independent of the effect of TSH on adenylate cyclase activity have also been demonstrated although the role of this phenomenon in the differentiation or function of the cell is not clear. Finally, TSH is known to have a trophic effect on thyroid cells. Thus, numerous studies have shown that the growth of functioning thyroid cells is accelerated by, or absolutely requires, the presence of TSH.
Graves' disease, an autoimmune thyroid disease characterized by primary hyperthyroidism, is sometimes indicated by the presence of a goiter. It was originally thought that Graves' disease was related to high blood levels of thyrotropin (TSH). Subsequent studies of blood samples taken from patients afflicted with Graves' disease indicated that TSH could not usually be found at elevated levels. The failure to detect small amounts of TSH may be due, in part, to the methods of assay used therefor. Thus, the radioimmunoassay technique which has been used for decades and continues to be in current use depends upon measurement of iodinated TSH in a displacement test; it does not measure bioactive TSH, but rather immunologically reactive TSH, i.e., TSH which reacts with an antibody directed at a number of its structural features rather than only those features important for bioactivity or function. This technique, which is dependent upon a very potent anti-TSH antibody, the iodinated label in TSH, the purity of the labeled TSH and the quality of the antibody, is also incapable of detecting low levels of TSH since, at best, the limits of assay sensitivity are only in the range of normal serum TSH levels.
Still other studies indicated that Graves' disease was associated with autoantibodies in the circulation which were capable of stimulating thyroid function. Two types of stimulating autoantibodies are now known, those that mimic the TSH regulatory controls over growth and those that mimic the regulatory control over adenylate cyclase activity (cAMP levels). In other autoimmune thyroid diseases, autoantibody thyroid factors which inhibit as well as stimulate thyroid function are postulated. In each case the prior history of the thyroid preparation, for example, specific patient characteristics or prior therapy, might well influence the particular autoimmune antibody or TSH response elicited.
The first assay method for Graves' disease immunoglobulins (GD IgGs) found in patient sera was known as long acting thyroid stimulator (LATS) assay. LATS is measured by an in vivo assay which involves administering the immunoglobulin fraction of the patient to a mouse whose thyroid has been injected with radioiodide. The presence of certain immunoglobulins causes a release of thyroid hormones from the radio-iodinated mouse gland. The shortcomings of the LATS assay primarily are that it is capable of giving positive results in only 40 to 50 percent of cases in which it is used; it does not measure a significant number of stimulators detected by measurement of immunoglobulin-enhanced adenylate cyclase activity in human membrane, slice or cell preparations; it requires approximately four to eight mice per single dose of IgG sample and as many as twenty-four for a full dose curve, each mouse requiring 1 to 2 weeks of preparation time and 1 day of assay time, and its activity does not correlate with the severity or extent of the clinical disease. The drawback that the LATS assay was not sensitive to all thyroid stimulating immunoglobulins in a patient led to the measurements of stimulating antibodies in human membrane cell or slice systems. The assumption on which these assays are based was that the fundamental autoantibody stimulating activity was its ability to stimulate adenylate cyclase activity. The cyclic AMP thus produced appeared to function as a "second messenger" in the respect that it further stimulated the cell to perform many of its routine functions, such as iodide uptake, production of thyroglobulin, the conversion of thyroglobulin to thyroid hormones, and the release of thyroid hormones into the blood. The assumption that all stimulators, whether TSH or an autoimmune antibody or IgG, would behave like TSH in stimulating adenylate cyclase activity led to the development of in vitro assays of elevated cAMP levels as a measure of all thyroid stimulators.
In the search for an accurate in vitro assay method, several human thyroid membrane, cell and slice preparations and techniques have been developed which rely on the similar stimulatory effects of the immunoglobulins, as compared to TSH, to elevate the amount of cyclic adenosine 3', 5'-monophosphate or cyclic adenosine monophosphate (cyclic AMP or cAMP) in these tissue preparations. Although the human slice or cell preparations, both of which are currently being used in Graves' disease assays, are capable of indirectly measuring a significant number of stimulators by determinations of immunoglobulin-enhanced adenylate cyclase activity and are, therefore, more sensitive to and have a higher positive correlation with the disease state, these techniques also suffer from certain shortcomings. Thus, human thyroid material is either not readily available or is only episodically available in most clinical settings and is not usually normal human tissue. The human material, even when available, also has a high degree of variability in response, a problem which may relate to the individualistic history of each tissue source. The thyroid slice and primary cell culture systems of assay also depend, to a large extent, on the individual skills, experience and subjective judgment of the analyst performing the assay, for example, the analyst's subjective assessment of what is "good" human thyroid tissue. In membranes in particular, the level of hormone stimulatable adenylate cyclase, the enzyme which produces cAMP, is present in only small amounts. Thyroid slices in particular cannot be preserved in any manner and must be used in a fresh condition.
Although a technique has been developed whereby primary cell cultures may be frozen and tests subsequently performed on aliquots withdrawn from the frozen culture, there are still certain inherent disadvantages with the technique. Human thyroid cells do not grow in vitro and, therefore, the number of tests which may be performed is limited by the quantity of primary cells obtained and the above variability problems. Secondly, after the cells are dispersed, a waiting period of from two to four days is required before the cells may be employed in an assay. Another problem associated with the primary culture system is that thyroid cells are contaminated with fibroblasts or other cells which can respond to TSH or autoimmune-antibodies by yielding other agents or prostaglandins which can, in turn, stimulate the cAMP response in the thyroid cell. The relationship of this activity to autoimmune thyroid disease is not yet clear.
It became apparent to scientists and physicians that to further define molecular and regulatory mechanisms of thyroid physiology and pathology and to permit the development of a rapid, accurate and easily conducted clinical determination and/or quantification of thyrotropin and of thyroid stimulatory or inhibitory factors, an improved in vitro system would be needed. The ideal system would, preferably, be one that demonstrates the properties and characteristics of a normal and non-malignant cell, established in long-term or permanent cultures, which could be maintained in a totally controlled or chemically defined environment and cloned to give rise to genetically homogeneous cell populations. Such cells would also possess unlimited growth capacity while maintaining the expression of the biochemical markers belonging to the differentiated cell type from which they originated. A primary requirement of the cell line or strain is that their growth rate be rapid enough so that large populations of cells could be raised and used in either large-scale experiments or numerous assays.
Early studies seemed to suggest that a cell line of epithelial cells derived from normal Fischer rat thyroid (FRT) cells offered promise in assays of TSH and immunoglobulins in Graves' disease. Although FRT cells are extremely sensitive to cholera toxin, the FRT line lacks differentiated thyroid functions, such as TSH stimulated thyroglobulin biosynthesis and iodide trapping as a result of a defective glycoprotein component of the TSH receptor. These cells thus appear to be non-functioning or de-differentiated in culture. The FRT cell strain was isolated initially in medium supplemented with 5 percent serum and TSH or dibutyryl cyclic AMP and was subsequently conditioned by culture on thyroid fibroblasts. At first they were able to produce thyroglobulin in primary cultures but after adaptation to growth in unconditioned medium lost the ability to concentrate iodide or to produce thyroglobulin. Epithelial FRT cells possess a TSH-stimulatable membrane adenylate cyclase and ADP ribosyltransferase activity which does indicate their thyroid origin.
Another thyroid cell line FRTL, reported widely in the literature, was isolated from primary cell suspensions obtained from the thyroid glands of NIH Fischer 344 inbred strain of rats. The FRTL cell line was isolated in medium supplemented with 0.5 percent calf serum and purified hormones. The FRTL cell line, purified by successive colonial isolations, was found to maintain highly differentiated features, such as secretion into the culture medium of physiological amounts of thyroglobulin and the concentration of iodide by 100-fold, after three years in continuous culture. They were observed to maintain the same biochemical and morphological characteristics which typify the primary cultures of the thyroid follicular cells immediately after their enzymatic release from the rat thyroid, (Ambesi-Impiombato et al, Proceedings of the National Academy of Science (USA), 77, pages 3455 to 3459 (1980)). However, this thyroid cell line demonstrates only a limited growth potential in vitro, the population doubling time being approximately 5 to 7 days in Coon's modified Ham's F-12 medium supplemented with 0.1 to 0.5 percent calf serum and six hormones at 37 degrees C. Indeed, the fact that normal cells have only a limited growth potential in vitro has been generally accepted as a rule (Impress and Hayflick, "The Limited In Vitro Life Time Of Human Diploid Cell Stains", Experimental Cell Research, 37, pages 614 to 636 (1965)). Thus, growth and irreversible de-differentiation appeared to be absolutely linked to each other in vitro. This was heretofore believed to be due to intrinsic properties of the differentiated cells. Another characteristic of this line which limits its usefulness in testing the "acute" effects of the addition of TSH is that withdrawal of TSH for more than 24 hours from the hormone mixture in which the cells are grown results in detachment of the cells from the surface of the culture dishes in which they are grown and their subsequent progressive death. The FRTL cell line is also unable to achieve growth to confluency, the cells existing only as isolated sparse cells. Finally, unlike the FRTL-5 strain, the FRTL cell line requires insulin as an absolute growth requirement.